Not Applicable
Not Applicable
This invention relates to the discovery that an intermediate, differentiated stage of pancreatic stem cells exist that can be propagated in a stable manner in successive serial passaging while maintaining insulin production in response to glucose. These cells are advantageous in that they are both expandable and stable in culture and can be driven to late stage development. This invention further provides for culturing techniques that select for these intermediate differentiated stage cells and selectively eliminates early or late stage pancreatic cells.
The mammalian pancreas develops from the embryonic foregut bud. As the embryonic buds grow, a ductal system develops by branching morphogenesis. After the ventral and dorsal anlage fuse, the new organ grows and matures into two interlocked structures, the exocrine system and the endocrine system. The majority of the pancreas is composed of acinar cells that produce digestive enzymes. The endocrine system includes xcex2-cells, which produce insulin, xcex1-cells, which produce glucagon, and xcex4-cells, which produce somatostatin. The endocrine cells are organized into clusters called islets.
Animal research has shown at least two mechanisms of xcex2-cell formation: neogenesis from ductal precursor cells and replication of mature xcex2-cells. Replication of differentiated xcex2-cells is maintained postnatally into adulthood. Replication of xcex2-cells is accelerated by an increased demand for insulin, for example, as a result of high glucose infusion, partial pancreatectomy, and during gestation. Under these conditions, xcex2-cells mass quickly increases through both cell hypertrophy (enlargement of volume of individual cells) and hyperplasia (increase in the number of xcex2-cells).
In Type I or insulin dependent diabetes mellitus (IDDM) there is a clear reduction in the number of xcex2-cells due to an autoimmune attack against the xcex2-cells. Eisenbarth, N. Eng. J. Med. 314:1360-1368 (1986). A treatment for Type I diabetes could include increasing in the number of xcex2-cells in a subject suffering from Type I diabetes. Bonner-Weir, Endocrin. 141:1926-1929 (2000).
Another treatment for diabetes using islet cells involves grafting pancreatic tissue from immune matched donors into transplant recipients. Typically, transplant recipients are required to receive immunosuppressant therapy to prevent rejection of the transplanted organ. Recently developed immunosuppressant regimens have improved the results of clinical islet transplantation in humans. While the technique remains experimental, if islet cell transplants can perform the same function as whole organ pancreas grafts, this much simpler surgical procedure would play an important role in the treatment of diabetes.
Although the transplantation of human islets shows promise as a powerful treatment for diabetes, a number of impediments exist that presently limit the utility of this procedure. One significant impediment is the inability to produce sufficient numbers of islet cells for use in the procedure. Presently, the process used to obtain islets for transplantation typically involves isolation of pancreatic tissue, enzymatic digestion of the pancreatic tissue to liberate the individual cells from the surrounding tissue, and the use of a gradient centrifugation purification technique. The gradient centrifugation purification technique is well known in the art and is performed by many islet transplant centers. Unfortunately, the yield of islets from a single pancreas treated with the standard procedure is usually insufficient for transplantation. Accordingly, alternatives to this procedure have been sought and developed. The use of fetal tissue or xenogenic transplant tissue has been explored, but ethical issues, availability of source material, and concerns over immune rejection or xenotropic pathogenesis complicate such approaches.
To date the ability to isolate, culture, and expand pancreatic cells for use in transplantation to treat pancreatic endocrine disease has remained elusive. Although islet and islet cells can be isolated from pancreatic tissue, this isolated material remains viable and capable of endocrine function for only a short period of time if it is not properly preserved. Various approaches to isolating pancreatic stem cells and inducing differentiation in vitro have been reported (see Peck et al., Ann Med 33:186-192 (2001); Bonner-Weir et al., Proc Natl Acad Sci USA 14:7999-8004 (2000); U.S. Pat. Nos. 6,001,647; 5,928,942; 5,888,916; PCT publications WO 00/78929 and WO 00/47721). Previous methodologies, however, have suffered from several limitations. Expansion of the pancreatic cell population following isolation has generally required a period of growth in serum-containing medium (see. e.g., U.S. Pat. No. 5,888,916), which raises cost and safety issues. Moreover, while satisfactory cell proliferation is achieved by such methods, the resulting cell populations may not retain markers of pancreatic cell differentiation or the ability to produce insulin, and often cannot be consistently differentiated into viable and high hormone-producing cells.
Serum-free selective media, which can promote the growth of epithelial cell populations over less desirable cell types (see Stephan et al., Endocrinology 140:5841-54 (1999)), offer the possibility of overcoming some of these limitations. Serum-free culture conditions have been reported for culture of pancreatic cells isolated from adult tissues. See Bonner-Weir et al., supra; WO 00/78929. However, these procedures are not completely satisfactory. A period of culture in serum-containing medium, requiring special culture substrates, is still obligatory, and transition of the cells to a serum-free medium for differentiation eliminates their ability to propagate. What is required is a general method to isolate and culture pancreatic cellular material that consistently yields cells capable of proliferation in vitro while retaining the potential to produce pancreatic hormones. Such cell populations could reverse the diabetic state following transplantation, as well as serve as a source for pancreatic endocrine hormones in vitro, and provide model systems for the study of pancreatic development and disease. The present invention fulfills these and other needs.
This invention provides methods and compositions for culturing pancreatic cells in vitro. In one aspect, the invention provides a method of preparing a cell culture of propagating pancreatic cells, the method comprising the steps of isolating propagating pancreatic cells, transferring the cells to an epithelial-selective culture medium containing growth hormone and less than 1% total volume of serum, and culturing the cells through at least one passage in culture. This method yields a cell population capable of being expanded from about 180 cells per square centimeter to about 1,800 cells per square centimeter, which is characterized by the following properties: at least 90% of the cells are positive for the transcription factor PDX-1/IPF-1, and the insulin:actin mRNA ratio of the population is between 1:100 and 1000:1. In certain embodiments of the invention, at least 95%, 98%, 99%, or 100% of the cells stain positive for PDX-1. In one embodiment of the invention, the cells are capable of being expanded from about 90 cells per square centimeter and expanded to about 36,000 cells per square centimeter. In other embodiments, the insulin:actin mRNA ratio is between 1:10 and 100:1. In some embodiments of the invention, the insulin:actin mRNA ratio is the unstimulated level of insulin mRNA.
In one embodiment of the invention, prior to transfer of the cells to a culture medium containing less than 1% serum, the cells are maintained in a medium containing serum at between 1% and 4% of the medium volume. In another embodiment, the cells are maintained in a medium containing 4% or more serum by volume. The maintenance period in some embodiments is less than 24 hours, while in other embodiments the maintenance period is a number of days such as any number of days between one and 14 days. In some embodiments of the invention, transfer of the cells from a medium containing more than 1% serum or more than 4% serum occurs gradually, with successive transfers of the cells to medium containing lower and lower amounts of serum. In other embodiments, transfer of cells from medium containing more than 1% serum or more than 4% serum is accomplished in a single step, by a single medium change or serial passage. In one embodiment of the invention, the isolated pancreatic cells have a mixture of PDX-1 positive and PDX-1 negative phenotypes, and the propagation of the cells in medium containing less than 1% serum selectively propagates the PDX-1 positive cells.
In another aspect, the invention provides a method of maturing pancreatic cells into more differentiated cells that express high levels of endocrine hormones. This method comprises the steps of culturing pancreatic cells on a substrate to condition the substrate, removing the cells from the substrate, and reseeding pancreatic cells on the substrate, yielding an aggregate of pancreatic cells comprising an encapsulating mantle of cytokeratin-19 positive cells surrounding an inner cell mass, wherein the aggregate comprises 50-5000 cells and has a diameter of between 50 and 300 microns. In one embodiment, at least one cell of the inner cell mass stains positive for a marker of endocrine development selected from the group consisting of PDX-1, insulin, glucagon, somatostatin, and KS1/4. In other embodiments, a greater proportion of the inner cell mass, such as at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the cells stain positive for a marker of endocrine development. In one embodiment, at least one of the culturing steps to produce the aggregate takes place in a medium containing growth hormone and less than 1% serum by volume. In other embodiments, the starting culture of pancreatic cells is at least 90% PDX-1 positive and has an insulin:actin mRNA ratio of between 1:100 and 1000:1.
In another aspect, the invention provides a method of providing pancreatic endocrine function to a mammal, the method comprising the steps of culturing pancreatic cells on a substrate, removing the cells from the substrate, reseeding pancreatic cells on the substrate, allowing the cells to form aggregates with an encapsulating mantle of ck-19 positive cells and an inner cell mass, and implanting the aggregate within the mammal to provide pancreatic endocrine function.
In yet another aspect, the invention provides a culture of propagating pancreatic cells, having the ability to be passaged from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter, while retaining the properties of at least 90% of the cells staining positive for PDX-1 and the population insulin:actin mRNA ratio being between 1:100 and 1000:1.
In another aspect, the invention provides an aggregate of cultured pancreatic cells, comprising a surrounding mantle of ck-19 positive cells and an inner cell mass, wherein the aggregate comprises 50-5000 pancreatic cells and has a diameter of between about 50 and 300 microns.
xe2x80x9cAggregatexe2x80x9d in the context of cells refers to a three dimensional structure.
xe2x80x9cCK-19xe2x80x9d is a 40 Kd acidic keratin, cytokeratin 19.
xe2x80x9cInsulin:actin mRNAxe2x80x9d ratios are measured by band density using gel scanner or by real time PCR using different labels for insulin and actin (see example 3). It is an average across a population of cells.
xe2x80x9cImplantingxe2x80x9d is the grafting or placement of the cells into a recipient. It includes encapsulated cells and non-encapsulated for example in an alginate matrix. The cells can be placed subcutaneously, intramuscularly, intraportally or interperitoneally by methods known in the art.
xe2x80x9cPassagexe2x80x9d of cells growing as a monolayer attached to a surface usually refers to a transition of a seeded culture container from a partially confluent state to a confluent state, at which point they are removed from the culture container and reseeded in a culture container at a lower density. However, cells may be passaged prior to reaching confluence. Passage typically results in expansion of the cell population as they grow to reach confluence. The expansion of the cell population depends on the initial seeding density but is typically a 1 to 10, 1 to 5, 1 to 3, or 1 to 2 fold expansion. Thus, passaging generally requires that the cells be capable of a plurality of cell divisions in culture.
A xe2x80x9cpopulationxe2x80x9d of cells refers to a plurality of cells obtained by a particular isolation or culture procedure. While the selection processes of the present invention yield populations with relatively uniform properties, a population of cells may be heterogenous when assayed for marker expression or other phenotype. Properties of a cell population are generally defined by a percentage of individual cells having the particular property (e.g., the percentage of cells staining positive for a particular marker) or the bulk average value of the property when measured over the entire population (e.g., the amount of mRNA in a lysate made from a cell population).
xe2x80x9c90% PDX-1 positivexe2x80x9d refers to a statistical sampling of randomly selected cells. Standard immunochemistry techniques are used and positively stained cells are counted visually under a microscope. Percentage is determined by comparison with appropriately controlled samples, i.e., preparing identical cells and using an antibody of similar isotype but not specific for PDX-1.
xe2x80x9cSerumxe2x80x9d refers to material obtained from blood other than blood cells. Serum is typically obtained by clotting or by physical separation of blood cells by centrifugation and defibrination. As used herein, serum may be functionally defined by its biological activity: serum generally supports the growth of mammalian cells in culture when added to culture media. Serum may be obtained from a variety of species (e.g., human, bovine, ovine, equine, porcine, rabbit, chicken, and the like) and developmental stages (e.g., fetus, juvenile, or adult). In certain embodiments, xe2x80x9cserumxe2x80x9d also refers to serum supplement or replacement products obtained from fractionated serum or other sources, e.g. Select Soytone (Becton Dickinson) or other commercially available products. Such serum equivalents may be completely or partially defined.
xe2x80x9cMantlexe2x80x9d refers to an envelope of cells surrounding in three dimensions the inner cell mass.